2.1 Data Collection and Preprocessing
Expression data (HTSeq-FPKM) and matching clinical data for OSCC were obtained from TCGA (https://portal.gdc.cancer.gov). Those without RNA sequencing data and survival data were omitted. In our study, 379 OSCC samples were obtained from TCGA datasets was used to analyze LINC00937.
For the expression data, we converted HTSeq-FPKM data into Transcripts Per Million (TPM) reads and normalized by log2 (TPM + 1) for subsequent analysis.
2.2 Correlation Assessment of Immune Cell Infiltration
Immune infiltration data for several cells, such as B, CD4+ T, CD8+ T, dendritic, macrophages, and neutrophils, were acquired from the tumor immune estimation resource (TIMER) database (https://cistrome.shinyapps.io/timer/). Pearson's correlation determined correlations between immune infiltration levels and risk scores, while Spearman correlation was used to assess the association between immune cell infiltrations and lncRNA LINC00937.
2.3 Enrichment Analyses
Metascape (https://metascape.org) is a web-based tool for functional gene annotation as well as enrichment analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for enrichment analyses via the database for prediction of the biological significance of LINC00937 co-expression genes.
2.4 Protein-Protein Interactions Network
Search Tool for the Retrieval of Interacting Genes (STRING, https://string-db.org/) is an online bioinformatics tool for the prediction of functional associations among proteins. An interaction score not more than 0.4 was the cut-off threshold.
2.5 Patients and specimens
Ten pairs of OSCC patients with established OSCC diagnosis were collected by the Department of Oral and Maxillofacial Surgery, The Second affiliation Hospital of Harbin Medical University. In this study, none of the patients was treated with neoadjuvant therapy. Their OSCC samples and matched non-carcinoma tissue samples were first formalin-fixed, paraffin-embedded utilized for testing the expression of LINC00937. The Ethical Committees of the Second affiliation Hospital of Harbin medical university (KY2021-120) permitted our study. Each tissue was provided the informed consent before participation.
2.6 Fluorescence in situ hybridization (FISH)
FISH was used to test the expression of LINC00937 by a Probe Mix kit (Servicebio, Wuhan, China). OSCC tissue and normal tissue were firstly fixed in 4% paraformaldehyde (Solarbio, China) for ten minutes. The protocol was used previously. Then, the pre-hybridization pad was discarded, and 150 µl of the hybridization buffer was added with the lncRNA FISH Probe Mix. Hybridization was done at 37°C overnight, followed by washing with different buffers at 42°C. Nuclei were treatment with DAPI (Servicebio, Wuhan, China) and images collected by inverted fluorescence microscopy (Phenix, Jiangxi, China) in five random areas.
2.7 Cell Culture and siRNA Transfection
CAL-27, were obtained from the ShangCheng Beina ChuangLian Biology Technology Co., Ltd (Shanghai, China). OSCC cells were grown in DMEM (Gibco, United States) with 10% fetal bovine serum (Gibco, Australia) at 37◦C with a 5% CO2 incubator. LINC00937 siRNAs were obtained from RiboBIO (Guangzhou, China). siRNA sequences were:
si-h-LINC00937_001: 5-GAGGAATAACTTCACTCTT-3;
si-h-LINC00937_002: 5-GTATAAATTGAGCTGACT-3;
si-h-LINC00937_003: 5-GAGCTGACTGCAAGGTACT-3;
The LINC00937-siRNA targeting si-LINC00937 or negative control si-NC by RiboFECT™ CP (RiboBIO, Guangzhou, China). Control siRNAs were standard as the negative control.
2.8 RNA Isolation and Quantitative Real-Time qPCR
Total RNA was harvested and used for cDNA synthesis by Trizol reagent (Thermo Fisher Scientific, USA) and SuperScript II first-strand cDNA synthesis kit (Thermo Fisher Scientific, USA). RT-qPCR was detected by SYBR Premix Ex Taq (TaKaRa, China) based on the control. The 2 −ΔΔCt approach was used to determine expression levels relative to those of GAPDH. The primer sequences were as follow:
LINC00937
Forward: 5’-CGGGTCCTTCCTCTTCCCCA-3’;
Reverse: 5’-CGCAGCCTCTTCTCTTCGGG-3’
GAPDH
Forward: 5’-GAAGGTGAAGGTCGGAGTC-3’;
Reverse: 5’-GAAGATGGTGATGGGATTTC-3’
2.9 Cell growth and proliferation
CCK-8 (Cell Counting Kit 8, Servicebio, Wuhan, China ) and colony formation analyses were employed to test the cell growth. For the CCK-8 comments, OSCC cells were first inoculated in 96-well plates with cell numbers of 6000/well. The CCK-8 reagent was added to every well at varying time points (0, 24, 48, and 72 h), after which cells were stored at 37◦C for 2–4 hrs. All the cells were tested at the optical density (OD) of 450 nm by a microplate reader (Thermo Fisher Scientific, USA).
For colony formation assays, cells after treatment as before. After 14 days treatment cells crucial with 1% crystal violet Dissolve in methanol for 15–30 mins.
2.10 Trans-well assay
8-µm pore size chambers employed cell migration tests without the Matrigel gel. Cells with the number at 50000 were inoculated into the upper section with a none-serum media. At the lower well stage, 20% FBS-supplemented medium was added. After one day of incubation, all cells in the wells were stained, after which optical microscopy (Phenix, Jiangxi, China) was performed for observation. Five random areas were collected for each sample. Chambers also performed cell invasion assays with Matrigel gel. Other procedures were performed as earlier described.
2.11 Western blotting
Total protein of OSCC cells was isolated by using RIPA lysis buffer (Beyotime, Beijing, China) and separated on 12.5% SDS-gels before being transferred to polyvinylidene fluoride membranes (PVDF, Schwalbach, Germany). Following blocking with 5% non-fat milk for 2 hours at room temperature, the membrane was incubated overnight at 4°C. Anti-FGR (1:1000, Cell signaling technology, USA), anti-IL10AR (1:1000, Cell signaling technology, USA), as well as anti-GAPDH (1:10000, 8884, Cell signaling technology, USA) were used in our study. Incubation with horseradish peroxidase-labeled secondary antibody for 1 hour at room temperature was then performed. A chemiluminescence detection system was used to detect the bands. The loading control was GAPDH.
2.12 Statistical Analysis
R software (Version 3.6.1) analyzed statistical data and visualizations. Wilcox rank-sum test was applied when comparing differences between two groups, and the Kruskal-Wallis rank-sum tested when comparing differences among three groups. Kaplan-Meier analysis was for survival analysis between two groups of patients. The discrimination ability of lncRNA LINC00937 and hub gene in OSCC was assessed by receiver operating characteristic (ROC) analysis via the pROC package. The student's t-test evaluated Between-group differences. Data are shown as mean ± SD (n = 3), with P < 0.05 being significant.